Methods for assessing 1,25(OH)2 D3 activity in skin and for enhancing the therapeutic use of 1,25(OH)2 D3

ABSTRACT

Described are in vivo methods for testing the activity of vitamin D 3  (1,25-dihydroxyvitamin D 3 ) and analogs thereof in human skin and for testing the effect of a test compound on said activity. Also described are methods for enhancing the in vivo activity of vitamin D 3  and analogs thereof by coadministering vitamin D 3  or an analog thereof with an RXR ligand, a 24-hydroxylase inhibitor, or a mixture thereof. Preferred RXR ligand materials are retinoids, such as 9-cis retinoic acid, and precursors thereof, such as all trans retinoic acid. Preferred 24-hydroxylase inhibitors are imidazoles, especially those inhibiting cytochrome-P450 oxidative enzymes, such as ketoconazole. Also included is a method for determining whether a candidate compound inhibits 24-hydroxylase activity or induction thereof.

This application claims priority from provisional application number60/015,145, filed Apr. 5, 1996, the disclosure of which is incorporatedherein by reference.

TECHNICAL FIELD

This invention relates especially to the molecular action of1,25-dihydroxyvitamin D3 (1,25(OH)₂ D₃). More particularly it relates toassays for determining vitamin D activity in skin, to methods forenhancing the therapeutic use of 1,25(OH)₂ D₃ (and its analogs), and forincreasing the safety and/or effectiveness of using 1,25(OH)₂ D₃ intreatments for skin conditions.

BACKGROUND

Vitamin D3 was discovered as a result of efforts to determine thenutritive factor responsible for the antirachitic property (i.e.,ability to prevent rickets) of cod liver oil. Vitamin D3 (also calledcholecalciferol) is photosynthesized from 7-dehydroxycholesterol(previtamin D3) in skin by UV-induced cleavage of the carbon-carbon bondbetween C9 and C10, enters circulation, and binds to vitamin D bindingprotein (DBP) for transport. DBP-bound vitamin D3 is biologically inertand requires activation. In the liver vitamin D3 is hydroxylated at theC-25 position by a cytochrome P-450 enzyme system to monohydroxyvitaminD₃, 25(OH)D₃, the major circulating form of vitamin D. This metaboliteis hydroxylated again in the kidney at the C-1 position to formdihydroxyvitamin D₃, 1,25(OH)₂ D₃, the hormonally active vitamin Dmetabolite. Dihydroxyvitamin D₃ is metabolized by 24-hydroxylase at theC-24 position to form 1,24,25-trihydroxyvitamin D3 (1,24,25(OH)₃ D₃),which is biologically inert. The trihydroxyvitamin D₃ is ultimatelymetabolized to calcitroic acid. In vitro studies of the metabolism of1,25(OH)₂ D₃ indicate that it catalyzes its own catabolism, but amechanism therefor has not been suggested. Ray, S., et al., J. CellBiochem (1995) 59:117-122.

The cellular receptor for 1,25(OH)₂ D₃ (designated VDR, for Vitamin DReceptor) is a member of family II of the hormone receptor superfamilyof transcription factors. VDR has been fully characterized and isprimarily localized in the nuclear compartment of the cell. In the cellnucleus, VDR, in the presence of 1,25(OH)₂ D₃, heterodimerizes with theretinoid X receptor (RXR). This dimeric complex binds to a vitamin Dresponsive element (VDRE, characterized by direct repeats of the hexamerAGGTCA spaced by three nucleotides) and activates vitamin D responsivegenes.

9-cis retinoic acid is the native ligand for RXR. Its effect in vivo onthe above-described intracellular action of 1,25(OH)₂ D₃ is not known.

1,25(OH)₂ D₃ 's antirachitic activity is based on its enhancement ofintestinal absorption of dietary calcium and its mobilization of calciumstores from bone. More recently it has been found to decreaseproliferation and increase terminal differentiation of humankeratinocytes, and to exhibit immunosuppressive activity on Th₁ cells.These findings have led to its use to treat dermatological disorderssuch as psoriasis. The use of 1,25(OH)₂ D₃ to treat dermatologicaldisorders is tempered, however, by its potent calcemic effects. Thoseeffects have led to the development of 1,25(OH)₂ D₃ analogs, such ascalcipotriene, that have reduced calcemic effects. Even so, the searchcontinues for ways to improve the therapeutic efficacy of 1,25(OH)₂ D₃and its analogs in dermatology.

SUMMARY OF THE INVENTION

The present invention provides novel methods for assessing the 1,25(OH)₂D₃ activity of a test compound, and whether a test compound can enhancethe activity of 1,25(OH)₂ D₃ (or an active analog thereof). It alsoprovides methods for improving the therapeutic efficacy of 1,25(OH)₂ D₃and its analogs.

The present invention is primarily based on two scientific findings. Thefirst is that the 24-hydroxylase gene, which contains a VDRE, is presentin skin and is activated by 1,25(OH)₂ D₃ in vivo. Thus, indicia of suchactivation, such as 24-hydroxylase mRNA induction and 24-hydroxylaseproduction, are sensitive markers of vitamin D activity in vivo. Thisfinding has also led to the discovery of this invention, that24-hydroxylase inhibitors can increase vitamin D activity in vivo. Thesecond is that the RXR ligand enhances the intracellular action of1,25(OH)₂ D₃ in vivo.

Accordingly, one aspect of the present invention is an in vivo methodfor determining whether a candidate (test) compound exhibits 1,25(OH)₂D₃ activity in skin, which method comprises applying the test compoundto living skin, determining the amount of 24-hydroxylase mRNA or24-hydroxylase in the skin following the topical application of the testcompound, and comparing the concentration determined (of mRNA or thehydroxylase) to a control; an increase in the amount of 24-hydroxylasemRNA or 24-hydroxylase in the skin relative to the control indicates thecompound has 1,25(OH)₂ D₃ activity in skin.

Another aspect of the invention is an in vivo method for determining theeffect of a candidate (test) compound on the activity of 1,25(OH)₂ D₃ oran analog thereof (or mixtures thereof) in skin (i.e., in vivo) whichcomprises applying 1,25(OH)₂ D₃ or an analog thereof (or mixturesthereof) both with and without coapplication of the test compound toseparate areas of living skin, determining the amount of 24-hydroxylasemRNA or 24-hydroxylase in each of the separate skin areas following theapplication of the compound, and comparing the amounts determined; alarger amount of 24-hydroxylase mRNA or 24-hydroxylase in the skin towhich both 1,25(OH)₂ D₃ (or said analog) and the test compound wereapplied indicates that the test compound enhances the activity of1,25(OH)₂ D₃ (or the analog) in living skin.

Yet another aspect of the invention is a method of treating adermatological disorder characterized at least in part byhyperproliferation or hypodifferentiation of keratinocytes, or ofinflammation, in a human patient, which comprises topicallycoadministering to the area of skin of the patient affected by saiddisorder an amount of (a) 1,25(OH)₂ D₃ (or an analog thereof) and (b) atleast one of an RXR ligand and a 24-hydroxylase inhibitor, wherein thecombined amount is effective to ameliorate one or more dermatologicalsymptoms of the disorder. In preferred embodiments, the method comprisesapply 1,25(OH)₂ D₃, an RXR ligand, and a 24-hydroxylase inhibitor.

In yet another aspect this invention provides a method or improving thesafety and/or effectiveness of using an amount of 1,25(OH)₂ D₃ effectivefor the treatment of a dermatological disorder, which comprises reducingthe amount of 1,25(OH)₂ D₃ applied and administering therewith at leastone of an RXR ligand and a 24-hydroxylase inhibitor. By decreasing theamount of dihydroxyvitamin D₃ required in such treatments, hypercalcemiaand other undesirable effects thereof can be reduced because less of thevitamin is administered, thus improving the safety of such treatment.Viewed another way, a lesser amount of dihydroxyvitamin D₃ can be usedwhen co-administered with an RXR ligand and/or a 24-hydroxylaseinhibitor because the latter essentially increase the effectiveness ofthe lesser amount of the dihydroxyvitamin D₃ applied.

In still another aspect, the invention provides a method for determiningwhether a candidate compound inhibits 24-hydroxylase activity, whichcomprises inducing 24-hydroxylase activity in vivo in skin, biopsyingskin cells in which the 24-hydroxylase was induced, dividing thebiopsied cells into two groups, adding a tagged monohydroxyvitamin D₃ toa first group of cells and thereafter measuring the amount of taggeddihydroxyvitamin D₃, adding a tagged monohydroxyvitamin D₃ plus thecandidate compound to a second group of cells and thereafter measuringthe amount of tagged dihydroxyvitamin D₃, wherein a lesser amount oftagged dihydroxyvitamin D₃ in the second group tested indicatesinhibition of 24-hydroxylase activity or induction thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-8 are depict result of testing described in the examplesfollowing in the specification.

DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

As used herein the term "1,25(OH)₂ D₃ activity in skin" intends theability to decrease proliferation and/or increase terminaldifferentiation of human keratinocytes.

As used herein the term "1,25(OH)₂ D₃ analog" intends a compound thatpossesses 1,25(OH)₂ D₃ activity. Such analogs generally are structurallyrelated to 1,25(OH)₂ D₃, such as, without limitation, calcipotriene, andthose disclosed in U.S. Pats. Nos. 5,087,619, 5,145,846, 5,342,833,5,403,832, 5,428,029 and 5,451,574, EPA Pub. No. 0398217B1, and PCT Pub.No. WO 95/01960, the disclosures of which are incorporated herein byreference.

As used herein, terms such as "co-application", "co-administering", andthe like are intended to mean that the compounds are applied to the skinat relative times such that their actions are cooperative; preferably,the compounds are applied with a few hours of each other, morepreferably the compounds are applied sequentially, and most preferablythe compounds are applied simultaneously (e.g., in the same physicalformulation).

As indicated above the invention contemplates in vivo methods forscreening candidate compounds to determine whether such compounds (a)possess 1,25(OH)₂ D₃ activity in skin and (b) enhance 1,25(OH)₂ D₃activity in skin.

When screening a compound to determine whether it possesses 1,25(OH)₂ D₃activity in skin, the compound is first applied topically to skin.Normally the compound will be applied in the form of an ointment orlotion at one or more concentrations (usually between 0.001% to 0.1% byweight). A predetermined volume of the formulation is applied to apredetermined area of skin of normal healthy adults. The applicationsite(s) is occluded with an appropriate dressing for a predeterminedtime period, usually 2 to 4 days. Biopsy samples of the treated skin anduntreated (control) skin are then taken and processed to obtain nuclearor protein extracts, and the extracts can be subjected to Northern blotor biochemical enzyme activity analysis (e.g., as described below) todetermine its 24-hydroxylase mRNA amount (concentration) or24-hydroxylase activity.

When screening a compound to determine whether it enhances 1,25(OH)₂ D₃activity in skin, the compound is co-applied to the skin with 1,25(OH)₂D₃ or an analog thereof as above. 1,25(OH)₂ D₃ (or analog) willtypically be applied at a concentration between about 0.001% to 0.1% byweight. Skin treated only with 1,25(OH)₂ D₃ (or analog) is used as acontrol (i.e., without co-application). After occluding the skin areasto which application of the compound(s) was made, the 24-hydroxylasemRNA concentration or 24-hydroxylase activity of the treated skin isdetermined as described above.

The 24-hydroxylase mRNA content and 24-hydroxylase activity of normal,untreated skin is barely detectable. Various examples followingillustrate the levels of elevation in 1,25(OH)₂ D₃ that result fromtreating skin with a combination of the dihydroxyvitamin D₃ and a24-hydroxylase inhibitor.

Examples of 24-hydroxylase inhibitors are ketoconazole and otherdermatologically acceptable azoles or imidazoles that inhibitcytochrome-P450 oxidative enzymes. Still further, analytical methodsdescribed below explain both testing for 24-hydroxylase activity (or24-hydroxylase mRNA concentration) and testing for whether a particulartest compound inhibits the activity (whether the mode of inhibition isdirect or through inhibiting the transduction pathway).

As indicated above, the invention also contemplates using RXR ligandsinstead of and in addition to 24-hydroxylase inhibitors to increase theactivity of 1,25(OH)₂ D₃ or analogs thereof in living skin. Such ligandsand/or inhibitors are coadministered with 1,25(OH)₂ D₃ or an analogthereof When an analog is used in combination with a 24-hydroxylaseinhibitor in practicing the present methods, the analog must be asubstrate for 24-hydroxylase (e.g., the analog must be metabolized by24-hydroxylase into an inactive metabolite).

Examples of RXR ligand materials (also termed "RXR ligands" herein) thatare suitable for used the methods of this invention include, withoutlimitation, 9-cis retinoic acid and the RXR ligands described in PCTPubs. Nos WO 93/03713, WO 93/21146, WO 94/15902 and WO 95/04036, thedisclosures of which are incorporated herein by reference. Precursorsthat result in 9-cis retinoic acid (e.g. all trans retinoic acid) mayalso be used as the RXR ligand material.

The 1,25(OH)₂ D₃ or analog and the RXR ligand and/or 24-hydroxylaseinhibitor can be formulated for topical administration to skin eitherseparately or combined together in a mixture. As used herein, the term"topical" denotes the use of 1,25(OH)₂ D₃ /analog, RXRligand/24-hydroxylase inhibitor, incorporated in an acceptabledermatological carrier, and applied at the affected skin site for theexertion of local action.

Accordingly, the topical compositions include those pharmaceutical formsin which the compound is applied externally by direct contact with theskin. The topical dosage preparations comprise gels, creams, lotions,ointments, powders, aerosols, soaps, and other conventional forms forapplying medication to the skin by admixing the active ingredients withknown pharmaceutical topical carrier materials and adjuvants. Ointmentsand creams encompass formulations having oleaginous, absorbable,water-soluble, and emulsion-type bases such as petrolatum, lanolin,polyethylene glycols and the like. Lotions are liquid preparations andvary from simple solutions to aqueous or hydroalcoholic preparationscontaining finely divided substances. Lotions typically containsuspending or dispersing agents, such as cellulose derivatives (ethylcellulose, methyl cellulose, and the like), gelatin, or gums, andincorporate the active ingredient(s) in a vehicle of water, alcohol,glycerin and the like. Gels are semi-solid preparations made by gellinga solution or suspension of the active ingredients in a carrier vehicle.The vehicles, which can be hydrous or anhydrous, are gelled using agelling agent, such as, carboxypolymethylene, and neutralized to aproper gel consistency with the use of alkaline compounds, such assodium hydroxide and amines. Soaps (such as NIZORAL® brand ofketoconazole, provided as a 2% cream and a 2% shampoo) can be formulatedas solids (such as bars) or a liquids (such as shampoo) includingconventionally used surfactants, foaming agents, fragrances, and thelike. All topically applied compositions can include one or morecompatible adjuvants conventionally used in topically appliedcompositions, such as colorants, fragrances, humectants, demulcents, andthe like, and mixtures thereof that are compatible with each other aswell as with the active ingredients.

The therapeutic method of the invention may be used to treat skindiseases associated with hyperproliferation or hypodifferentiation ofepidermal cells or inflammation such as psoriasis, actinic keratoses(precancerous lesions typically caused by chronic UV exposure),disorders of keratinization such as ichthyoses and keratoses, andsebaceous gland diseases such as acne or seborrheic dermatitis. In thesemethods, the 1,25(OH)₂ D₃ or analog will normally be administered dailytopically in formulations having concentrations ranging between 0.002%to about 0.05% (by weight) of the dihydroxyvitamin D₃. Correlatively,the RXR ligand will normally be coadministered at concentrations ofabout 0.025% to 0.1% (depending upon potency) and the 24-hydroxylaseinhibitor is coadministered at about 1% to 3% (depending on potency)daily. Thus, it should be understood that one aspect of this inventionis enhancing the treatment of a dermatological condition which istreated with high doses of 1,25(OH)₂ D₃ by decreasing the effectiveamount of dihydroxyvitamin D₃ administered and coadministering therewitha 24-hydroxylase inhibitor, an RXR ligand, or a combination of both(i.e., a composition comprising dihydroxyvitamin D₃, a 24-hydroxylaseinhibitor, and an RXR ligand). The coadministration of the inhibitorand/or the ligand provides a safer treatment, by reducing the effectiveamount of dihydroxyvitamin D₃ required to treat the condition, therebyreducing potential hypercalcemia, and provides a more effectivetreatment, by enabling the use of a lower dose of the dihydroxyvitaminD₃.

The methods of this invention are further illustrated by the followingexamples. These examples are not intended to limit the invention in anymanner. Following, "24-OHase" means `24-hydroxylase`.

EXAMPLE 1

Effect On 1,25(OH)₂ D₃ in Skin of 9-Cis Retinoic Acid TopicalApplication

Solutions of 1,25(OH)₂ D₃ (0.002%, 0.05%), 9-cis retinoic acid (0.1%)and 1,25(OH)₂ D₃ plus 9-cis retinoic acid (0.002% plus 0.1%) wereprepared in a vehicle of 95% ethanol in propylene glycol (7:3 v/v)containing 0.5 mg butylated hydroxytoluene per ml of solution.

One hundred microliters (μl) of the test solutions were applied to 3×6cm areas on buttock skin of normal healthy adults. Treatment sites wereoccluded with Saran brand plastic wrap, covered with a light-proofdressing and secured with surgical tape for 2 days. Epidermal biopsieswere taken using a keratome device with a blade set at 0.2 mm to cutnear the junction of epidermis-dermis. Thus, biopsies containedprimarily epidermis with residual amount of dermis (approx. 10%maximum). Biopsies were snap-frozen immediately in liquid nitrogen.Total RNA was then isolated from the biopsies as described by Elder etal., Retinoic acid receptor gene expression in human skin, J. InvestDermatol 96(4):425-33 (1991). 15 μg RNA from each isolate was subjectedto Northern blotting analysis as described in Xiao et al., in "Cloning,expression, and transcriptional properties of the human enhancer factorTEF-1", Cell 65(4):551-68 (1991). Human 24-hydroxylase cDNA fragments(Chen et al., "Isolation and expression of human 1,25-dihydroxyvitaminD3 24-hydroxylase cDNA", Proc Natl Acad Sci USA 90(10):4543-7 (1993))were labeled with α-[³² P]-dCTP (3000 Ci/mmole, DuPont-NEN, Boston,Mass.) using a random priming kit purchased from Boehringer Mannheim(Indianapolis, Ind.).

FIG. 1 is a graph depicting the results of the Northern blots. In FIG.1, the following abbreviations are used: VEH=vehicle; 9cRA=9-cisretinoic acid; D3 1,25(OH)₂ D₃ ; 24-hydroxylase=24-OHase. As shown inFIG. 1, 0.002% D3 increased 24-OHase mRNA induction by 11% whereas 9cRAby itself yielded no significant increase. However, addition of 9cRA to0.002% D3 together caused a synergistic 125% increase (relative to D3alone) in 24-OHase mRNA induction.

EXAMPLE 2

Effect on 1.25(OH)₂ D₃ In Skin of 24-Hydroxylase Inhibitor TopicalApplication Solutions of 1,25(OH)₂ D₃ (0.002%, 0.05%), ketoconazole(I%), and 1,25(OH)₂ D₃ plus ketoconazole (0.002%+1%) were prepared asdescribed above.

Skin was treated with these solutions and analyzed for 24-hydroxylasemRNA induction as described above. The results of these tests are shownin FIG. 2, in which Keto=ketoconazole. As shown in that Figure, D3 aloneincreased 24-OHase mRNA induction by approximately I 1% whereasketoconazole treatment yielded no significant increase. However, thecombination of D3 and keto acted synergistically to give a 320% increase(relative to D3 alone) in 24-OHase mRNA induction.

EXAMPLE 3

Determination of Effect on 1,25(OH)₂ D₃ Activity in Skin of all transRetinoic Acid

All trans retinoic acid is known to isomerize in skin to 9-cis retinoicacid. Solutions of D3, all trans retinoic acid (0.1%), and D3 plus alltrans retinoic acid (0.002% plus 0.1%) were prepared as above. Skin wastreated with those solutions and analyzed for 24-OHase mRNA induction asdescribed above. The results of these tests are shown in FIG. 3, wheretRA=all trans retinoic acid. As shown in FIG. 3, the combination of D3and tRA yielded an increase in 24-OHase mRNA induction greater thanwould be expected from the increase caused by D3 alone and tRA alone.

EXAMPLES 4-8

The substance of the following examples is also described in theinventors' paper entitled "The Retinoid X Receptor Agonist9-cis-Retinoic Acid and the 24-Hydroxylase Inhibitor KetoconazoleIncrease Activity of 1,25-Dihydroxyvitamin D₃ in Human Skin In Vivo" invol. 108 of J. Invest. Dermatol., 1997 (the disclosure of which isincorporated herein by reference).

Materials

1,25(OH)₂ D₃, 25-hydroxyvitamin D₃ or cholecalciferol [25OHD₃ ],24,25-dihydroxyvitamin D₃ [24,25(OH)₂ D₃ ], 25,26-dihydroxyvitamin D₃[25,26(OH)₂ D₃ ], 1,24,25(OH)₃ D₃, 1,25,26-trishydroxyvitamin D₃[1,25,26(OH)₃ D₃ ], and 9-c-RA were obtained from Hoffmann La Roche(Nutley, N.J.). All-trans-retinoic acid (t-RA) was obtained from Sigma(St. Louis, Mo.). Ketoconazole powder was obtained from Johnson andJohnson (Skillman, N.J.) and Janssen Pharmaceutical (Beerse, Belgium).Deoxycytidine 5'-[α-³² P]triphosphate and 25-[³ H]OHD₃(25-OH[26,27-methyl-³ H]cholecalciferol) were obtained from DuPont-NEN(Boston, Mass.) and Amersham Life Science (Arlington Heights, Ill.),respectively.

Treatment of Subjects and Procurement of Tissue

Solutions of 1,25(OH)₂ D₃ (0.0004%, 0.002%, 0.01%, and 0.05%), 9-c-RA(0.1%), t-RA (0.1%), ketoconazole (1%), 1,25(OH)₂ D₃ plus 9-c-RA (0.002%and 0.1%, respectively), 1,25(OH)₂ D₃ plus t-RA (0.002% and 0.1%,respectively), and 1,25(OH)₂ D₃ plus ketoconazole (0.002% and 1%,respectively) were prepared in a vehicle comprising 95% ethanol andpropylene glycol at 7:3 (vol/vol). Each solution also contained theanti-oxidant butylated hydroxytolulene (0.5 mg per ml). One hundredmicroliters of the study solutions were applied once to areas 3 cm×6 cmon buttock skin of normal healthy adults. Sites were occluded with Saranbrand wrap and then covered with a tape-secured light-proof dressing for2 days. For treatment with ketoconazole plus 1,25(OH)₂ D₃, the studysites were treated with the imidazole for 3 hours before application ofthe combination solution. Treated areas were infiltrated with 1%lidocaine local anesthesia and biopsied with a keratome (≈150 μm thick).Keratomed specimens were immediately frozen in a liquid nitrogen andstored at -70° C. until used.

Measurement of 24-OHase mRNA.

For measurement of 24-OHase mRNA, total cellular RNA was extracted fromkeratome biopsies obtained as described above. The guanidiniumisothionate-cesium chloride procedure was used as described (Elder etal, 1990). RNA species (20 μg per sample) were electrophoreticallysize-fractionated on agarose gels and transferred to derivatized nylonmembranes. The blots were sequentially hybridized against ³² P-labeled24-OHase cDNA and 36B4 (Astrom et al, 1991). DNA probes were prepared byrandom priming (Boehringer Mannheim, Indianapolis, Ind.) and quantitatedby using a PhosphorImager (Molecular Dynamics, Sunnyvale, Calif.) asdescribed (Elder et al, 1990; Astrom et al, 1991), except that 36B4rather than cyclophilin was used as the control probe. Integratedautoradiographic intensity obtained by PhosphorImager for the 24-OHasegene was first divided by that of the control gene (36B4). The divisionproduct was then divided again by that of the control treatment forrelative comparisons. Because the negative control (vehicle) groupcontained many small numbers, including zero, which precluded therequired computations (division by zero), data are expressed as percentinduction of the positive control (0.05% 1,25(OH)₂ D₃), which wasconsidered maximum induction.

Measurement of 24-OHase Enzyme Activity

Human skin treated with vehicle and 0.05% 1,25(OH)₂ D₃ for 2 days undercontinuous occlusion was keratome-biopsied. The biopsy specimens wereincubated in trypsin (0.1% in solution consisting of 30 mM HEPES buffer,10 mM glucose, 3 mM KCl, 130 mM NaCl, and 1 mM sodium phosphate, pH 7.2)for 30 min at 35° C. to separate the epidermis from the dermis.Dispersed epidermal cells were centrifuged at 2000×g for 5 min. Thepelleted cells were resuspended in 5 ml of 0.1% trypsin inhibitor andthen centrifuged again at 2000×g for 5 min. The cells were washed twicewith 5 ml of the digestion buffer (without trypsin). The final cellpellet was resuspended in 1 ml of enzyme assay buffer (20 mM HEPESbuffer, 125 mM KCl, 20 mM succinate, 2 mM MgCl₂, 1 mM dithiothreitol and1 mM ethylenediamine tetraacetic acid, pH 7.4). To 20 μl of resuspendedcells, 1 μM 25-[³ H]OHD₃ (1 μCi) was added to start the reaction. Whenused, ketoconazole was added at a final concentration of 1×10⁻⁴ M. Aftera 30-min incubation at 35° C., the reaction was stopped with theaddition of 2.6 ml of methanol:chloroform. 2:1 (vol/vol). After adding1.2 ml of chloroform and 1 ml of water, the samples were vortex-mixedfor 1 min and then allowed to separate into two layers. The aqueouslayer was washed twice with 1.2 ml of chloroform. With each wash, thechloroform layer was pooled and then evaporated to dryness, and thematerial was resuspended in 200 μl of methanol. The material wasfiltered with Millipore SJHV 004 filters into high-performance liquidchromatography vials. After evaporation to dryness, the material wastaken up in a final volume of 30 μl for high-performance liquidchromatography analysis.

High-Performance Liquid Chromatography Separation of Vitamin D₃Metabolites

The standard solutions used to identify and quantitate vitamin D₃metabolites were 25OHD₃, 24,25(OH)₂ D₃. 1,25(OH)₂ D₃, 25,26(OH)₂ D₃,1,24,25(OH)₃ D₃. and 1,25,26(OH)₃ D₃. Vitamin D3 metabolites wereseparated by using a Hewlett-Packard 1090M high-performance liquidchromatography system with a chem workstation, a pilot wavelength of 264nm, and a Spherisorb ODS1 column of 4.6 ×200 mm. An isocratic elutionwith 90% solvent A (acetonitrile) and 10% solvent B (0.005 M ammoniumacetate:acetic acid. 100.1 (vol/vol)) was achieved with a flow rate of0.7 ml/min at 26° C. for 22 minutes. The effluent from the column floweddirectly into a Radiomatic model 295A flow through liquid scintillationspectrometer for quantitation of tritium in each of the separatedmetabolites.

Statistical Analysis

Comparisons of mean levels of 24-OHase mRNA among treated sites weremade with either the paired t test or the repeated measures analysis ofvariance. All p values are two-sided. Summary statistics are expressedas means±SEM. Data was performed with the use of the MichiganInteractive Data System (MIDAS) a statistical software package developedat the Center for Statistical Consultation and Research at theUniversity of Michigan.

Results

24-OHase Gene Expression Is Induced by 1,25(OH)₂ D₃ in Human Skin

To determine whether the 24-OHase gene in human skin can be regulated invivo by 1,25(OH)₂ D₃, four different concentrations of the hormone wereapplied once under occlusion for 48 hours. Northern blot analysisrevealed a concentration-dependent induction of the 24-OHase mRNAlevels, as shown in FIG. 4. In vehicle treated skin, the mRNA level wasvery low and detectable. For this reason, the data are expressed asfractions of the level observed with 0.05% 1,25(OH)₂ D₃, the highestconcentration used, set arbitrarily at 100% (maximum induction). Ascompared with vehicle treatment, the application of 0.002% 1,25(OH)₂ D₃caused a small but statistically significant increase in the mRNA level.In the Northern Blot analysis shown in FIG. 4: * means p<0.05 versusvehicle (open bar); n=5; and the inset blots are from two representativesubjects.

24-OHase Gene Expression by 1,25(OH)₂ D₃ Is Synergistically Induced by9-c-RA

After determining the dose-response curve for 24-OHase mRNA induction by1,25(OH)₂ D₃ application, we investigated whether the small increase in24-OHase mRNA caused by a low dose of 1,25(OH)₂ D₃ (0.002%) could beaugmented by the application of 9-c-RA. Compared with vehicle, 9-c-RA byitself did not change human epidermal 24-OHase gene expression after 48h of occlusive treatment, as shown in FIG. 5. Similar to the resultsshown in FIG. 4, 0.002% 1,25(OH)₂ D₃ alone induced a small butsignificant increase in 24-OHase mRNA level (18% of maximum; p<0.05versus vehicle, n=15). Unexpectedly, however, when 0.002% 1,25(OH)₂ D₃was co-administered with 0.1% 9-c-PA, the combination caused asynergistic increase in mRNA levels to 33% of maximum. More particularlyin FIG. 5: *p<0.05 versus 0.002% 1,25(OH)₂ D₃ ; .sup.† p<0.05 versusvehicle; n=15; D₃ and 1,25(OH)₂ D₃ are represented by the strippedhatching. The combination treatment (0.002% D₃ plus 0.1% 9-c-RA) has abar height greater than the additive sum; the stippled hatching showsthe synergistic effect. Again, the inset blots are results from tworepresentative subjects.

24-OHase Gene Expression by 1,25(OH)₂ D₃ Is Synergistically Induced byt-RA

We have demonstrated that after topical application of t-RA to humanskin, its stereoisomers including 9-c-RA can be detected in the viableepidermis (Duell et al, 1996). Therefore, we next sought to determinewhether concomitant treatment of 1,25(OH)₂ D₃ with t-RA can enhanceinduction of 24-OHase mRNA, similar to that observed with 9-c-RAco-application. Two days of occlusive treatment with t-RA alone did notinduce 24-OHase mRNA expression, as compared with vehicle, as shown inFIG. 6. Compared to 0.05% 1,25(OH)₂ D₃ (maximum induction), 1,25(OH)₂ D₃enhanced the expression of 24-OHase mRNA by 28% (p<0.05 versus vehicle,n=15). In the presence of 0.1% t-RA, however, 0.002% 1,25(OH)₂ D₃treatment significantly induced the mRNA level to more than 40% of thelevel observed with 0.05% 1,25(OH)₂ D₃. In FIG. 6: *p<0.05 versus 0.002%1,25(OH)₂ D₃ ; ⁵⁵⁴ p<0.05 versus vehicle; n=15. As with FIG. 5, thestippled section of the combination treatment depicts the synergisticeffect of the combination therapy, and the insets are from tworepresentative subjects.

24-OHase Gene Expression by 1,25(OH)₂ D₃ Enhanced by Ketoconazole

Ketoconazole is a dermatologically suitable imidazole known to inhibitseveral P-450 enzymes involved in hormone metabolism. It also possessesan inhibitory activity against retinoic acid 4-OHase, which inactivatest-RA (Duell et al., 1992). Inhibition of 4-OHase by liarozole, a relatedimidazole, augments human skin responses to low doses of retinoic acidand retinol (Kang et al. 1996). Ketoconazole can similarly influencevitamin D signaling in human skin by inhibiting the 24-OHase. To supportthis, the induction of 24-OHase mRNA by 1,25(OH)₂ D₃ was examined in thepresence of and in the absence of the ketoconazole. Consistent withprevious examples presented herein, 0.002% 1,25(OH)₂ D₃ alone caused asmall but significant increase in the 24-OHase mRNA level as comparedwith vehicle (18% of maximum; p<0.05), as shown in FIG. 7. Ketoconazoleat 1% alone did not alter the mRNA level. Ketoconazole, when combinedwith 0.002% 1,25(OH)₂ D₃, however, synergistically increased thetranscription level to approximately half of the maximal inductioncaused by 0.05% 1,25(OH)₂ D₃, as shown in FIG. 7. In FIG. 7: *p<0.05 vs.0.002% 1,25(OH)₂ D₃ ; .sup.† p<0.05 vs. vehicle, n=5; again, thestippled pattern shows the enhanced benefits of combination therapyaccording to this invention.

1,25(OH)₂ D₃ Induces Human Epidermal 24-OHase Activity and KetoconazoleInhibits the Induced 24-OHase Activity

We studied the inducibility of the cytochrome P-450 enzyme activity inhuman skin in vivo by 0.05% 1,25(OH)₂ D₃ occlusive treatment for 2 daysto support the inhibition of 24-OHase by ketoconazole. Epidermal cellsderived from vehicle- and 1,25(OH)₂ D₃ -treated sites were assayed fortransformation of 25-OHD₃ to 24,25(OH)₂ D₃. Compared to vehicle (25±15pg per min per mg of protein), there was a 10-fold increase (p<0.05;n=5) in the formation of 24,25(OH)₂ D₃ by 1,25(OH)₂ D₃ (260±78 pg permin per mg of protein) as shown in FIG. 8. This induced activity of24-OHase by 1,25(OH)₂ D₃ treatment was completely inhibited by additionof ketoconazole (1×10⁻⁴ M) ex vivo, also shown in FIG. 8, in which theopen bar is vehicle, the striped bar is 1,25(OH)₂ D₃, and the solid baris 1,25(OH)₂ D₃ plus ketoconazole.

The concentration-dependent induction of 24-OHase mRNA expression by1,25(OH)₂ D₃ indicates that in human skin in vivo, the presence of onlythe VDR ligand is sufficient to activate the gene. The barely detectable24-OHase mRNA levels in normal (vehicle treated) skin and the robustinduction of this gene transcript by 1,25(OH)₂ D₃ make this a usefulbioassay to test for vitamin D activity of a compound in human skin invivo. This appears analogous to cellular retinoic acid binding protein(CRABP)-II mRNA induction by topical retinoid application to human skin.Enhanced expression of this gene, whose far upstream promoter contains aretinoic acid-responsive element (Astrom et al, 1991), by an occlusivetreatment with natural or synthetic retinoids has predicted theirretinoidal activity in human skin in vivo (Griffiths et al., 1993; Elderet al, 1995). The potential value of 24-OHase gene expression as ameasure of vitamin D activity in human skin, however, is greater thanthat for CRABP-II mRNA induction for retinoids. This is because,although retinoids typically give dose dependent responses clinically(i.e., erythema) and/or histologically (i.e., hyperplasia of theepidermis), 1,25(OH)₂ D₃ and its synthetic analog calcipotriene, whichis currently in clinical use, do neither. Thus, induction of 24-OHasemRNA provides a detectable 1,25(OH)₂ D₃ target in the absence of anyappreciable clinical or histologic activity in human skin in vivo.Furthermore, this enhanced expression of 24-OHase by 1,25(OH)₂ D₃treatment correlates with an increase in 24-OHase enzyme activity.

The ability of 9-c-RA to synergistically enhance the 24-OHase mRNA levelinduced by a low dose of 1,25(OH)₂ D₃ indicates that RXR is not a silentpartner in RXR-VDR heterodimer complex-mediated vitamin D signaling inhuman skin in vivo. In cell culture, human keratinocytes from normaladults transfected with a reporter gene VDRE (DR3)-tk-CAT (where CAT ischloramphenicol acetyltransferase) demonstrated more than a doubling ofchloramphenicol acetyltransferase activity when 9-c-RA was added to alow dose of 1,25(OH)₂ D₃. This chloramphenicol acetyltransferaseactivity could be further increased by over-expressing VDR and/or RXR,in the transfected keratinocytes, which strongly indicates the directcontribution of the receptors to the ligand-mediated inducibility.

Although 9-c-RA is the only natural ligand of RXR identified to date, itis also an agonist of RARs (Heyman et at, 1992; Allenby et al, 1993). Inaddition, because 9-c-RA can isomerize to t-RA in human skin tissue(Duell et al, 1996), a contribution of RAR-VDR in vitamin D signalingafter 9-c-RA and 1,25(OH)₂ D₃ co-treatment cannot be excluded. This isunlikely to be of major significance in human skin, however, because inepidermal nuclear extract preparations, we have not detected endogenousRAR-VDR heterodimers binding to VDREs. Furthermore, in our in vitrotransfection system and in C57BL/6 adult mice in vivo, the use ofRXR-specific ligand SR 11237 provided results that were comparable withthat of 9-c-RA, synergistically enhancing 24-OHase mRNA induction by1,25(OH)₂ D₃. The lack of adequate toxicology data to assure safe use inhumans prevented us from applying this synthetic compound to humans(hence its application to mice).

Similar to 9-c-RA, t-RA synergistically enhanced 24-OHase mRNA inductionwith a low dose (0.002%) of 1,25(OH)2D₃. Compared to 1,25(OH)₂ D₃treatment alone, addition of t-RA increased the transcription level by50%, and 9-c-RA did so by 76% (p=0.74 vs 50% increase). The comparablemagnitude of modest synergism caused by the two RA isomers with1,25(OH)₂ D₃ is not inconsistent with RXR-VDR being the majorheterodimer complex in vitamin D signaling in human skin in vivo. Wehave recently reported that the same 0.1% solution of t-RA applied tohuman skin for 48 h yielded approximately 7 nM 9-c-RA in the viableepidermis (Duell et al, 1996). Dissociation constant (Kd) valuesreported for 9-c-RA binding to RXRs have ranged from 11 to 18 nM (Heymanet at, 1992; Levin et al, 1992; Allenby et al, 1993). The Kddetermination made with endogenous retinoid receptors (RXR and combined)in nuclear extracts from human epidermis, in which the level of RXRprotein is 5-fold greater than that of RAR protein, was 1.2 nM (Fisheret al, 1994). Therefore, the 7 nM 9-c-RA that forms after 0.1% t-RA islikely sufficient to activate skin RXRs. Indeed, although 0.1% 9-c-RAtreatment results in 260 nM 9-c-RA in stratum corneum-free epidermis(Duell et al, 1996), no significant difference in synergism with1,25(OH)₂ D₃ was detected between 9-c-RA and t-RA (FIGS. 5-6).

Although both t-RA and 9-c-RA augmented vitamin D signaling,1,25(OH)2D₃, at concentrations used in our study, did not increase ordecrease the ability of t-RA or 9-c-RA to stimulate relevant targetgenes. When the blots were stripped of 24-OHase probes and reprobed withCRABP-II probes, we found, consistent with previous observations (Kanget al, 1995; Elder et al, 1996), that both t-RA and 9-c-PA markedlyinduced CRABP-II mRNA levels. Compared with a single treatment of t-RAor 9-c-RA, co-administration of each RA isomer with 1,25(OH)₂ D₃ did notinfluence the level of CRABP-II gene transcripts (data not shown).

In human skin in vivo, neither 9-c-RA nor t-RA alone induced 24-OHasemRNA expression. Similar results were obtained from cultured humankeratinocytes and mouse skin in vivo. By contrast, in mouse kidney,systemic administration of both natural and synthetic ligands enhancedthe expression of the 24-OHase gene without the presence of vitamin D3(Allegretto d al. 1995). This apparent tissue-specific gene regulationindicates that, in addition to the receptors and their cognate ligands,other factors are involved in the process. The importance of relativeratios of VDR, and RXR, in determining the degree of ligandresponsiveness (Lemon and Freeman, 1996) and the identity of the RXRisoform in influencing VDR heterodimer transactivation of the 24-OHasegene by 1,25(OH)₂ D₃ and 9-c-RA (Kephart et al. 1996) were recentlydemonstrated. It is also possible that as described for estrogen andthyroid hormone receptors (Chen and Evans, 1995; Horlein et al, 1995)co-activator and/or co-repressor proteins may be modulating VDR and RXRactivities. Therefore, tissue-restricted expression of thereceptors/receptor isoforms and/or co-activators/co-repressors may beresponsible in part, for the differential response to ligands observedin skin and kidney.

Ketoconazole is a broad-spectrum P-450 inhibitor that has been usedclinically in a variety of conditions ranging from fungal infections,seborrheic dermatitis, and psoriasis to prostatic carcinoma (Farr etal., 1985; Green et al, 1987; Mahler et al., 1993; Degreef andDeDoncker, 1994). Topically, ketoconazole has been safely used as a 2%cream and shampoo formulations. We found 1% ketoconazole to be effectivein synergistically increasing the 24-OHase mRNA level caused by a lowdose of 1,25(OH)₂ D₃. The marked inhibition of 1,25(OH)₂ D₃ -inducedhuman epidermal 24-OHase activity by ketoconazole ex vivo (FIG. 8)implies that the synergism in vivo was achieved by impeding theinactivation of 1,25(OH)₂ D₃ by topical ketoconazole. By itself,however, application of ketoconazole did not induce 24-OHase mRNAexpression. This is probably because, under in vivo conditions, aneffective inhibition of 24-OHase activity by topical ketoconazole doesnot increase endogenous 1,25(OH)₂ D₃ to a level efficient to induce24-OHase mRNA.

Our data indicate that vitamin D signaling in human skin in vivo can besignificantly increased by two different mechanisms: RXR ligands and24-OHase inhibitors. Although not specifically studied, RXR ligands willlikely increase vitamin D signaling by synthetic compounds, such ascalcipotriene, which can bind and transactivate target genes via VDR.Because the synergistic effects observed with 9-c-RA and ketoconazoleare mediated by distinct pathways, a combination of both will likelylead to enhancement of the bioactivity of 1,25(OH)₂ D₃. Combinationtopical therapy with RXR agonist and/or 24-OHase inhibitor allows theuse of less 1,25(OH)₂ D₃ than would be necessary when using 1,25(OH)₂ D₃as a single agent, to deliver a desired biologic/therapeutic response tohuman skin. In principle, a reduction in the amount of applied 1,25(OH)₂D₃ required to produce a pharmacologic effect would minimize potentialrisk of hypercalcemia.

While the present invention is described particularly with reference tohuman subjects, and includes in vivo evidence using human subjects,there are likely various veterinary utilities for the instant inventionfor other animals, especially mammals, exhibiting vitamin D metabolismanalogous to that in human skin.

As described herein, the assays and testing methods were performed invivo on human subjects (after informed consent). It should be understoodthat the invention herein is also applicable to methods performed onliving skin tissue cultures and on matrices supporting living skin cells(as are used in the development of artificial skin).

The foregoing description is meant to be illustrative and not limiting.Various changes, modifications, and additions may become apparent to theskilled artisan upon a perusal of this specification, and such are meantto be within the scope and spirit of the invention as defined by theclaims.

What is claimed is:
 1. A method of treating a dermatological disorderassociated with hyperproliferation or hypodifferentiation ofkeratinocytes of a human patient, which comprises topicallycoadministering to the area of skin of said patient affected by saiddisorder a first dermatologically suitable composition comprising1,25dihydroxy Vitamin D₃ (1,25(OH)₂ D₃) or an analog thereof, and a seconddermatologically suitable composition comprising a member selected fromthe group consisting of (a) a Retinoid X Receptor (RXR) ligand or aprecursor of an RXR ligand and (b) a 24-hydroxylase inhibitor, or amixture thereof, in a combined amount effective to treat saiddermatological disorder.
 2. The method of claim 1 wherein the RXR ligandis 9-cis retinoic acid and the precursor is all trans retinoic acid. 3.The method of claim 1, wherein said first and second dermatologicallysuitable compositions are provided together as a single composition. 4.The method of claim 1 wherein the 24-hydroxylase inhibitor isketoconazole.
 5. The method of claim 1, wherein the precursor is alltrans retinoic acid, and the 24-hydroxylase inhibitor is ketoconazole.6. A method for enhancing the activity of 1,25-dihydroxy Vitamin D₃(1,25(OH)₂ D₃) or an analog thereof in vivo in human skin,comprising:applying an effective amount of a dermatologically suitablecomposition comprising at least one member selected from (i) RXR ligandcompounds and precursors thereof and (ii) a 24-hydroxylase inhibitor, ora mixture thereof, and to the skin.
 7. The method of claim 6, whereinsaid RXR ligand is a retinoid.
 8. The method of claim 7, wherein saidfirst and second compositions are provided as a unitary mixture.
 9. Themethod of claim 6, the dermatologically suitable composition definedtherein being a first composition, and further comprising providing asecond dermatologically suitable composition comprising 1,25(OH)₂ D₃ oran analog thereof, and coadministering said second composition with saidfirst composition.
 10. The method of claim 9, wherein said retinoid isselected from the group consisting of retinoic acid and a precursorthereof.
 11. The method of claim 10, wherein the retinoid is9-cis-retinoic acid.
 12. The method of claim 10, wherein the retinoid isall trans-retinoic acid.
 13. The method of claim 6, wherein theinhibitor is ketoconazole.
 14. The method of claim 6, wherein saidactivity is enhanced in a method for treating a dermatological conditionby the topical application of 1,25(OH)₂ D₃.
 15. The method of claim 14,wherein the safety or effectiveness of said method for treating adermatological condition is enhanced using less 1,25(OH)₂ D₃ than wouldbe necessary when using 1,25(OH)₂ D₃ as a single agent bycoadministering said dermatologically suitable composition.
 16. Themethod of claim 6, wherein said composition comprises a mixture of (i)and (ii).
 17. The method of claim 7, wherein said composition comprisesa mixture of (i) and (ii).
 18. The method of claim 17, wherein saidunitary mixture, comprises 1,25(OH)₂ D₃, an RXR ligand or a precursorthereof and a 24-hydroxylase inhibitor.